This paper describes the partial purification and characterization of polygalacturonase and pectin lyase isolated from Carica papaya pericarp cv. solo 8...
Int.J.Curr.Microbiol.App.Sci (2017) 6(6): 2729-2739 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume Number (2017) pp 2729-2739 Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2017.606.326 Partial purification and Characterization of Two Pectinases (Polygalacturonase and Pectin lyase) from Papaya Pericarp (Carica papaya cv solo 8) Adingra Kouassi Martial-Didier1, Konan Kouassi Hubert2*, Kouadio Eugène Jean Parfait2, Yapi Jocelyn Constant2 and Tano Kablan1 Laboratoire de Biochimie Alimentaire et de Technologie des Produits Tropicaux de l’Université Nangui Abrogoua (Abidjan, Côte d’Ivoire), 02 BP 801 Abidjan 02, Côte d’Ivoire Laboratoire de Biocatalyse et des Bioprocédés de l’Université Nangui Abrogoua (Abidjan, Côte d’Ivoire), 02 BP 801 Abidjan 02, Côte d’Ivoire *Corresponding author ABSTRACT Keywords Biochemical properties, Carica papaya, Pericarp, Polygalacturonase, Pectin lyase, Purification Article Info Accepted: 26 May 2017 Available Online: 10 June 2017 Plant wastes could be a potential source of novel pectinases for use in various industrial applications due to their broad substrate specificity with high stability under extreme conditions Therefore, the application conditions of novel pectinases (Polygalacturonase and Pectin lyase) from Carica papaya cv solo pericarp were optimized in this study Those enzymes were partially purified by ammonium sulphate precipitation, dialysis and isoelectric focusing The polygalacturonase and pectin lyase were partially purified 1.3 and 1.26 fold with a yield approximately 20 % and 24 %, respectively, following purification with isoelectric focusing The specific activities of 3.62 and 0.176 UI/mg for polygalacturonase and pectin lyase enzymes were calculated, respectively The optimal pH of 5.0 and 8.0 were obtained for the polygalacturonase and pectin lyase, respectively while an optimal temperature of 45°C was obtained for polygalacturonase and 50 °C for pectin lyase Polygalacturonase and pectin lyase activities were enhanced by Na +, Ca2+, K+, and Mg2+ while EDTA has any effect at all concentrations on pectin lyase activity whereas EDTA was inhibitory to polygalacturonase activity The results suggest that Carica papaya pericarp peels can be used for value added synthesis of pectinase, an important enzyme with numerous biotechnological applications Introduction Pectinases are responsible for the degradation of the long and complex molecules called pectin that occur as structural polysaccharides in middle lamella and primary cell wall of higher plant Depending on their mode of action, the enzymes hydrolyzing pectin are broadly known as pectinases, which include pectin methylesterases (E.C.3.1.1.11), polygalacturonase (PG) (E.C.3.2.1.15) and pectin lyase (PL) (E.C.4.2.2.10) (Kashyap et al., 2001) PG cleaves the polygalacturonic acid backbone of the pectin and reduces the average length of the pectin chains (Baron and Thibault, 1985) The pectin lyases (PL) act on pectins, oligomers and polymers of galacturonic acid by catalyzing the rupture between two galacturonic acid units by a βelimination mechanism (Jeantet et al., 2007) Pectinases have been used in various processes and industrials, which in the 2729 Int.J.Curr.Microbiol.App.Sci (2017) 6(6): 2729-2739 degradation of pectic substances is essential (Favela-Torres et al., 2005) Polygalacturonase and pectin lyase have a great commercial significance in industrial application (Kashyap et al., 2001; Khan et al., 2013; Tu et al., 2013; Kumar and Suneetha, 2014) Whereas most industrial production of pectinases is limited to some species of bacteria, yeast (Gummadi and Panda, 2003; Jayani et al., 2005; Combo et al., 2012) Pectinase enzyme has been the focus of research for many years due to the potential and wide applications in various industrial processes An increasing demand of pectinase has rendered the need of exploring under product capable of producing novel pectinases with improved activities The screening realized by Yao (2013) on pectinases showed that their activities were two times higher in the pericarp than papaya pulp at mature stage presenting 1/8 yellow skin This pericarp (peel) could be valorizing to production of commercial pectinases This paper describes the partial purification and characterization of polygalacturonase and pectin lyase isolated from Carica papaya pericarp cv solo Materials and Methods Plant material and enzyme extraction The survey carried on the peel (pericarp) of papayas (Carica papaya cv solo 8) The ripe papayas are presenting 1/8 of the yellow skin studied in this work, were harvested from a farm near Thomasset (Azaguié), a village located at about 50 km of north of Felix Houphouet Boigny Airport, Abidjan (Côte d’Ivoire) The papaya pericarp (50 g) was ground using a blender in 50 ml NaCl solution 0.9 % (w/v) The homogenate was subjected to sonication (4 °C) at 50 - 60 Hz frequency using a BANDELIN SONOPLUS HD 2200 for 10 and then centrifuged at 10.000 g for 30 at °C The supernatant was filtered through cotton wool and was kept refrigerated and used as the crude extract Chemicals products Polygalacturonic acid, pectin from citrus fruits, bovine serum albumin (BSA) and Bradford reagent were supplied by SigmaAldrich (St Louis, MO, USA) The electrophoresis reagents on polyacrylamide gel were supplied by BIO-RAD All chemicals and reagents used were analytical grade Enzyme assay The polygalacturonase (PG) activity was determined by a standard colorimetric method of Miller (1959) using 3.5-dinitrosalicylic acid (DNS) 900 µl of subtract (0.5 % of polygalacturonic acid prepared in 100 mM of acetate buffer pH 5) was mixed with 100 µl of enzymatic extract for a total of 1000 µl Then the mixture was incubated at 40 °C for 15min The reaction was stopped by adding 1.5 ml of DNS After DNS was added the whole mixture was placed in a boiling water bath for Finally, the mixture was let to cool down at ambient temperature and the absorbance was read at 540 nm using a blank (mixture without subtract) and D-galacturonic acid for the standard curve The galacturonase activity was expressed as the equivalent of galacturonic acid produced per milligram of protein per minute (µmoL/mg/min) The pectin lyase (PL) activity was determined using the method of Preiss and Ashwell (1963) An assay mixture (2.5 ml) consisting of a 100 mM Tris-HCl buffer pH 8.0, 10 mM CaCl2, 0.5 % pectin and enzyme solution was incubated at 40 °C for h After incubation, the activity was determined by measuring the absorbance of the reaction mixture at 548 nm 2730 Int.J.Curr.Microbiol.App.Sci (2017) 6(6): 2729-2739 One unit of pectin lyase activity was defined as the amount of enzyme present in ml of enzymatic solution which released μM galacturonic acid for Protein estimation Protein concentrations and elution profiles from chromatographic columns were determined by Bradford method (Bradford, 1976) Bovine serum albumin (BSA) was used as the standard protein Partial purification of enzymes The Partial purification procedure was carried out in the cold room (4°C) Fifteen (15) ml of the enzymatic crude extract of the papaya (Carica papaya L cv solo 8) pericarp were saturated with different concentrations of ammonium sulphate (20-80 %) and left with gentle stirring for 24 h in a refrigerator at 4°C The mixture was then centrifuged at 6000 g for 30 at 4°C with a refrigerated centrifuge (MIKRO 22R, Hettich) The precipitated proteins contained in the pellet were suspended either in ml acetate buffer (0.1 M pH 5) or ml Tris-HCl buffer (0.1 M pH 8) respectively for PG and PL The fractions containing the pectinases activities are pooled and subjected to dialysis The dialysis extracts were immersed in a large volume of buffer (0.1 M acetate pH 7.5 or 0.1 M Tris-HCl pH 7.5) with continuous stirring for 16 h The membrane having pores allowed small molecules such as ammonium and sulfate ions to cross, while not allowing the large protein molecules to pass through it Every hours the buffer solution was renewed This step made it possible to remove the excess of salt but also the substances of low molecular weight such as the pigments contained in the enzymatic raw extract The dialyzed extracts were subjected to an isoelectric-focusing technique To start balancing the ion exchange membranes with a solution of phosphoric acid and sodium hydroxide overnight before using the membranes This operation consisted in immersing the red anode in a solution of H3PO4 (0.1 M), the black cathode in a solution of NaOH (0.1 M) Then the focusing chamber was prepared This preparation consisted of mixing 100 μl of dialyzed extract, 150 μl ampholyte (Bio-lyte® 3/10, Bio-Rad Laboratories Hercules) and 2.75 ml of distilled water Fill the ml syringe with the sample and slowly charge the focus chamber Then, ml of 0.1 M H3PO4 and ml of 0.1 M NaOH were respectively added to the ventilation hole of the anode (red button) and cathodic (black button) assembly Finally, the isoelectric focusing was carried out at 350 V, mA, 25 W for h Once the IEF race was completed, the fractions were harvested as quickly as possible to avoid the diffusion of the separated proteins PG and PL activities were assayed in each fraction to identify the isoelectric point of enzyme isolated from papaya pericarp cv solo Electrophoresis of proteins The partial purity of the enzymes was analyzed by native-polyacrylamide gel electrophoresis (Native-PAGE) (Laemmli, 1970) Silver staining was employed to identify protein bands Enzyme characterization Effect of pH The influence of pH on the activity of purified polygalacturonase and pectin lyase were probed at different pH, either in 0.1 M sodium acetate buffer (pH 3.5 - 5.5), in 0.1 M sodium phosphate buffer (pH 5.5-7.5) or in 0.1 M Tris-HCl buffer (pH 7.0-9.0) PG and PL 2731 Int.J.Curr.Microbiol.App.Sci (2017) 6(6): 2729-2739 activities were determined under standard conditions describe above The system without any additive was used as a control pH stability of enzyme Results and Discussion To determine the pH stability, the polygalacturonase and pectin lyase were preincubated in 0.1 M acetate buffer (pH 3.55.5) and 0.1 M Tris-HCl buffer (pH 7.0-9.0) respectively at °C for h Then assayed for polygalacturonase and pectin lyase activities in 0.1M acetate buffer (pH 5) and 0.1 M TrisHCl buffer (pH 7.5) respectively Partial purification of polygalacturonase and pectin lyase Effect of temperature The effect of temperature on polygalacturonase and pectin lyase activities was determined by varying the temperature from 30 to 80 °C at an interval of °C The PG and PL activities were assayed as decribed above using polygalacturonic acid and pectin as substrates Determination of energy of activation and Q10 values The energy of activation (Ea) was estimated from the slope of Arrhenius plot obtained by plotting the Log of initial enzyme velocity against the reciprocal of the absolute temperature (Riet and Tramper, 1991) Initial velocities were determined at different temperatures under standard assay conditions Q10 values were determined according to the method of Segel (1975) Influence of metallic ions and EDTA The effects of metallic ions (Na+, K+, Ca2+, Mg2+, Ba2+) and EDTA were determined by adding into the reaction system individually with the final concentration of mM, 10 mM and 15 mM The residual activity was determined under the standard condition as A summary of the steps utilized to purify partially the pectinases from papaya cv solo pericarp was recorded in table The process of pectinases partial purification was achieved with a protocol consisting of three steps The crude pectinases were partially purified by ammonium sulphate precipitation, dialysis and isoelectric focusing This first step ammonium sulphate precipitation 30 % not only concentrates the pectinases but also to eliminate some of the pigments contained in the raw extract (data not shown) The specific activities of polygalacturonase and pectin lyase in the step were evaluated as 2.4 UI/mg and 0.11 UI/mg, respectively (Table 1) Niharika and Abhishek (2014) also used 30 % ammonium sulphate precipitation as the first step for purification of apple pomace pectinases Hammed and Alyaa (2010) used also ammonium sulphate precipitation as the first step for the purification of pectin lyase of Erwinia carotovora isolated from the spoilt potatoes After ammonium sulphate precipitation the second step was dialysis The increase in the specific activity of polygalacturonase (2.8 UI/mg) and pectin lyase (0.14 UI/mg) after dialysis could be explained by a loss of undesired proteins which interfere with the activity of pectinases (Undenwobele et al., 2014) These two first steps of purification (ammonium sulphate precipitation and dialysis) of enzymes have already been reported by Ahmed et al., (2016) in the purification of vegetable pectinases The last step in the purification (Isoelectric focusing) of the polygalacturonase and pectin lyase isolated from the papaya pericarp cv solo made it possible to improve the 2732 Int.J.Curr.Microbiol.App.Sci (2017) 6(6): 2729-2739 purification factor but also to determine the isoelectric point of the enzyme This step made it possible to improve the specific activity of polygalacturonase (3.62 UI/mg) and pectin lyase (0.17 UI/mg) Polygalacturonase and pectin lyase were partially purified about 1.3 and 1.26 fold with final proteins of 2.86 mg and 3.56 mg, respectively (Fig 1) The specific activity of papaya pericarp cv solo polygalacturonase (3.62 UI/mg protein) obtained was less than polygalacturonase of Achaetomium sp Xz8 (28.122 UI/mg protein) (Tu et al., 2013) and polygalacturonase of Aspergillus niger MTCC 3323 (54.3 UI/mg protein) (Arotupin et al., 2012) Otherwise, specific activity of papaya pericarp cv solo pectin lyase obtained was also less than pectin lyase of Bacillus subtilis BPLSY1 (58.85 UI/mg protein) (Al Balaa et al., 2014) These differences in specific pectinases activities could be explained by the nature of the biological material used, by the technique used for purification and but also because these enzymes were partially purified Partial purities of polygalacturonase and pectin lyase were verified by performing native polyacrylamide gel electrophoresis This electrophoresis shows a single protein task for polygalacturonase and pectin lyase but we remark a few protein tasks in band (Figure 2) This result indicates that those enzymes were partially purified Characterization of polygalacturonase and pectin lyase The results obtained demonstrated the maximum activities of polygalacturonase and pectin lyase partially isolated from papaya pericarp were observed at pH 5.0 and pH 8.0, respectively Maximum polygalacturonase activity at pH 5.0 proves the acidophilic nature of polygalacturonase The optimum pH of our findings was similar to the pectinase of Penicillium varidictum RFC3 and Aspergillus niger (Silva et al., 2002; Fahmy et al., 2008), respectively The range of pH at which polygalacturonase activity is maximal is between pH 3.5 and 6.0 (Fenghour et al., 2002; Amande et al., 2013) Polygalacturonase optimum pH was higher than that of purified polygalacturonase tomato (pH 4.4) (Verlent et al., 2004) and lower than polygalacturonase of kiwi (pH 5.5) (Shouqing, 2014), Aspergillus niger and Aspergillus flavus (pH 5.5) (Deshmukh et al., 2012) and Bacillus sp KSM-P 410 (pH 7) (Jayani et al., 2005) Polygalacturonase from Carica papaya pericarp could be favorable for the treatment in acid medium such as papaya juice (pH 5.6) (Tu et al., 2013) Whereas maximum pectin lyase activity at pH 8.0 confirms the alkalophilic nature of pectin lyase partially purified The optimum pH of the pectin lyase of the papaya pericarp CV solo is identical to that of Aspergillus niger isolated from orange peel (Batool et al., 2013) and Aspergillus flavus (Yadav et al., 2008) On the other hand, it is lower than the optimum pH (pH9.5) of pectin lyase isolated from Bacillus subtilis BPLSY1 (Al Balaa et al., 2014) Polygalacturonase and pectin lyase activities were stable between pH 4.5 and 5.5 in sodium acetate buffer and pH ranging from pH 7.0 and 9.0, respectively This pH range is almost similar to that of the purified polygalacturonase of Penicillium sp CGMCC 1669 which is between pH and pH (Yuan et al., 2011) These results suggest that this pH range (4.5-5.5) would be favorable for the expression of the enzyme This zone could be ideal for clarification of fruit juices which have acidic pH, for the maceration of fruit and vegetables that have acidic pH Concerning pectin lyase pH stability, these results suggest that working on this enzymatic activity in a pH range between and 9; Could be ideal for treatment whose pH was in a basic medium 2733 Int.J.Curr.Microbiol.App.Sci (2017) 6(6): 2729-2739 Table.1 Partial purification procedure of polygalacturonase and Pectin lyase from papaya pericarp cv Solo Purification steps Crude extract PG PL (NH4)2SO4 precipitation PG PL Dialysis PG PL Isoelecric focusing PG PL Total protein (mg) Total activity (Units) Specific activity (Units/mg) Yield (%) Purification fold 326.93 326.93 680 32 2.08 0.098 100 100 1 85.8 81.26 206 9.25 2.4 0.114 30.33 28.9 1.15 1.16 17.72 17.90 49.56 2.5 2.8 0.140 24.06 27.03 1.17 1.23 2.86 3.52 10.35 0.62 3.62 0.176 20.88 24.8 1.3 1.26 Table.2 Some physicochemical characteristics of polygalacturonase and pectin lyase from papaya pericarp cv Solo Values given are the averages of at least three experiments ± SE Physicochemical properties Optimum temperature (°C) Optimum pH pH stability Activation energy (kJ/mol) Q10 Activator agents Inhibitor agents Values PG 45 5.0 4.5-5.5 77.12 ± 0.03 2.67± 0.1 Mg2+, Ca2+, K+, Na+ Ba2+, EDTAa PL 50 8.0 7.5-9 68.77 ± 0.05 1.61± 0.1 Mg2+, Ca2+ Ba2+, K+, Na+ - Values given are the averages of at least three experiments ± SE a: ethylene diamine tetraacetic acid Fig.1 Effect of different ammonium sulphate saturation on polygalacturonase and Pectin lyase from papaya pericarp 2734 Int.J.Curr.Microbiol.App.Sci (2017) 6(6): 2729-2739 Fig.2 Native-PAGE of papaya pericarp Lane 1, crude extract; lane 2, Polygalacturonase purified and lane 3, pectin lyase purified The highest PG and PL activities were observed at 45 °C and 50 °C, respectively Papaya pericarp polygalacturonase optimum temperature obtained in this work was in agreement with the polygalacturonase produced by gamma irradiated Penicillium citrinum (El- Batal et al., 2013) This optimum temperature was lower than that of tomato polygalacturonase (Verlent et al., 2004) Indeed, these authors obtained an optimum temperature of 55 °C In addition partial purified Carica papaya pericarp pectin lyase optimal temperature was 50°C This value was lower than pectin lyase from mango peel optimum temperature (60°C) (Amande et al., 2013) From these results, polygalacturonase and pectin lyase isolated from Carica papaya could be used in food industry for processes requiring moderate temperatures Those optimum temperatures (45°C and 50°C) were recommended for clarification and depectination operations This treatment must be carried at temperatures between 45 and 50 °C to avoid gelling during clarification operations (Grampp, 1977) These mesophilic enzymes could be coveted in the food industry during thermal operations (Table 2) During thermal activation, polygalacturonase activation energy (Ea) and pectin lyase Ea were 77.12 ± 0.03 kJ/ mol and 68.77 ± 0.05 kJ/mol (Figure 4C and 4D) with Q10 values of 2.67 and 1.67, respectively According to Lee and Wiley (1970) more Ea was high more the reaction was held quickly The activation energy (Ea) value of the polygalacturonase of Carica papaya pericarp was four times lower than the value founded by Benen et al., (2003) which is 324.17 kJ/mol on tomato polygacturonase of variety CXD 199 These results suggest that the polygalacturonase isolated from Carica papaya pericarp cv solo 2735 Int.J.Curr.Microbiol.App.Sci (2017) 6(6): 2729-2739 reacted less rapidly than polygalacturonase isolated from tomato variety CXD 199 The activities of polygalacturonase and pectin lyase partially purifies were highly activated by Mg2+, Na+, K+ and Ca2+ (Figure 5) This result is agreement with Rexova-Benkova (1976) who reported that generally pectinases were activated by the presence of cations The activation of pectin lyase activity by the Ca2+ was already been reported by several authors (Ren and Kermode, 2000; Arotupin et al., 2012; Pedrolli and Carmona, 2014) According to Al Balaa et al., (2014), in absence of the calcium ion (Ca2+) the pectin lyase activity was zero Whereas, the polygalacturonase activity from Carica papaya pericarp was inhibited by 10 mM and 15 mM Ba2+ However, the result was in contrary to pectinase enzyme from fruit and vegetable wastes and Aspergillus niger strain MCAS2, that Ba2+ ions found to enhance the pectinase activity (Ramachandran and Kurup, 2013; Khatri et al., 2015) This suggests that the requirement of metals ions for the polygalacturonase activity vary depending upon their sources In addition the inhibitory effect of barium ion would probably be a result of interactions between cations and peptide chains and not directly on the enzyme (Walter, 1991) Then EDTA was an inhibitor of PG activity but had no impact on the pectin lyase activity Our results were similar to those of Arotupin et al., (2012) These authors confirmed the inhibitory of EDTA in Aspergillus niger CSTRF polygalacturonase On the other hand, our results are different from those of Whitaker (1984) showed that EDTA was generally an inhibitor of pectin lyase activity due to Ca2+ chelation In this study, the partial purification and some characteristics properties of polygalacturonase and pectin lyase extract from Carica papaya cv solo pericarp were investigated The polygalacturonase and pectin lyase were partially purified successfully and showed reasonable stability to different conditions From this characterization of papaya pericarp polygalacturonase and pectin lyase, it may be a potential candidate for industrial uses Therefore, it can be employed in industries for hydrolysis of pectic biomass to utilizable bio-product Future studies on pectinases isolated from Carica papaya pericarp should be devoted to the understanding the effect of heat treatment on these enzymes Acknowledgments The authors would like to acknowledge University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca, Laboratory of Molecular Nutrition and Proteomics, Institute of Life Sciences, Romania for providing technical support for this work This research was supported by “Eugen Ionescu” PhD research scholarship of the Romania Ministry of Foreign Affairs References Ahmed, I., M A., Zia, M A., Hussain, Z., Akram, M T., Naveed, and Nowrouzi 2016 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Kouassi Hubert, Kouadio Eugène Jean Parfait, Yapi Jocelyn Constant and Tano Kablan 2017 Partial Purification and Characterization of Two Pectinases (Polygalacturonase and Pectin Lyase) from Papaya Pericarp (Carica papaya cv solo 8) Int.J.Curr.Microbiol.App.Sci 6(6): 2729-2739 doi: https://doi.org/10.20546/ijcmas.2017.606.326 2739 ... Jocelyn Constant and Tano Kablan 2017 Partial Purification and Characterization of Two Pectinases (Polygalacturonase and Pectin Lyase) from Papaya Pericarp (Carica papaya cv solo 8) Int.J.Curr.Microbiol.App.Sci... from Carica papaya pericarp cv solo Materials and Methods Plant material and enzyme extraction The survey carried on the peel (pericarp) of papayas (Carica papaya cv solo 8) The ripe papayas are... the partial purification and some characteristics properties of polygalacturonase and pectin lyase extract from Carica papaya cv solo pericarp were investigated The polygalacturonase and pectin